The unique properties of nanoparticles can be exploited for delivering DNA into cells. Among the nanoparticles investigated (e.g., tungsten, aluminum, nickel, etc.), Gold NanoParticles (GNP) tend to be excellent candidates for delivery of DNA. The low cytotoxicity and ease of functionalization with various ligands of biological significance make gold nanoparticles a preferential choice for transformation. Gold nanoparticles can range in size from 1.2 nm-600 nm. The commonly used synthesis of GNP produces a negatively charged (e.g., citrate coating) surface for particles from 20-400 nm, whereas smaller 1-10 nm range of GNPs are positively charged. Plasmid DNA, which is sufficiently flexible to partially uncoil its bases, can be exposed to gold nanoparticles. In the case of the citrate-functionalized GNP, the plasmid DNA can partially uncoil. The negative charges on the DNA backbone are sufficiently distant so that attractive van der Waals forces between the bases and the gold nanoparticle cause plasmid DNA to be attached and coat the surface of the gold particle. Whereas, in the case of the positively charged GNP, electrostatic and van der Waals forces can contribute to coating or attachment of the DNA.
In addition to metal nanoparticles, semi-conductor nanoparticles (e.g., quantum dots) (“QD”) within the size range of 3-5 nm have also been used as carriers to deliver molecules into cells. DNA and proteins can be coated or linked to the QD surface that is multifunctionalized with a ligand (see, e.g., Patolsky et al. (2003) J. Am. Chem. Soc. 125:13918). Carboxylic acid or amine multifunctionalized QDs can be cross-linked to molecules containing a thiol group (see, e.g., Dubertret et al. (2002) Science 298:1759; Akerman et al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99:12617; Mitchell et al. (1999) J. Am. Chem. Soc. 121:8122), or an N-hydroxysuccinimyl (NHS) ester group, by using standard bioconjugation protocols (see, e.g., Pinaud et al. (2004) J. Am. Chem. Soc. 126:6115; Bruchez et al. (1998) Science 281:2013). An alternative way is to multifunctionalize QDs via conjugation with streptavidin. The streptavidin conjugates with biotinylated proteins, oligos, or antibodies (see, e.g., Dahan et al. (2003) Science 302:442; Pinaud et al. (2004), supra; Wu et al. (2003) Nature Biotechnol. 21:41; Jaiswal et al. (2003) Nature Biotechnol. 21:47; and Mansson et al. (2004) Biochem. Biophys. Res. Commun. 314:529.
Nanoparticles have been used to deliver plasmid DNA to a variety of animal cells. It has been found that when DNA-coated nanoparticles are incubated with cells not having a cell wall, the cells take up the nanoparticles and begin expressing any genes encoded on the DNA. Where nanoparticle delivery to cells normally having a cell wall is desired, the cell wall is stripped before the addition of the particles to protoplasts (see, e.g., Torney et al. (2007) Nature Nanotechnol. 2:295-300). In plant cells, the cell wall acts as a barrier for the delivery of exogenously applied molecules. Many invasive methods, like the gene gun (biolistics), microinjection, electroporation, and Agrobacterium, have been employed to achieve gene and small molecule delivery into these walled plant cells. Delivering small molecules and proteins across the cell wall and into the plant cell would be advantageous for the development of enabling technologies for the in vitro and in vivo manipulation of cells, tissues, and organs of intact plants.
Although well established in bacteria, yeast, animal cells, and moss, gene addition (i.e., the introduction of foreign DNA into a predetermined genomic location) remains a significant challenge in higher plants. Site-specific transgene integration occurs at a very low frequency in plant cells as compared to random integration, even when the incoming DNA contains large stretches of sequence homologous to host DNA (Halfter et al. (1992) Mol. Gen. Genet. 231(2):186-93; Lee et al. (1990) Plant Cell 2(5):415-25; Miao and Lam (1995) Plant J. 7(2):359-65. For example, a highly efficient Agrobacterium-based transfection system and herbicide selection resulted in gene targeting frequencies of up to 5×10−4 in rice. Attempts to enhance gene targeting efficiencies in plants have included the use of negative selection markers, and the use of plants genetically engineered to exhibit higher targeting frequencies. These efforts notwithstanding, random DNA integration via non-homologous processes continue to be a major impediment to gene targeting in plants. Given the general utility envisioned for targeted gene addition in the modification of crops for agricultural and industrial biotechnology, a solution to this problem is sorely needed.
In this regard, substantial increases in the frequency of gene targeting in a broad range of plant and animal model systems have been observed following the induction of a DNA double-strand break (DSB) at a specific genomic location in host cells, which stimulates a native cellular process, homology-directed DSB repair. Naturally occurring site-specific endonucleases whose recognition sites are rare in the plant genome have been used in this manner to drive transgene integration into a target sequence previously transferred into the plant genome via random integration. These studies highlight the potential of targeted DSB induction to stimulate gene targeting in plant cells, though the challenge of introducing a DSB in a native locus remains.
In animal cells, the solution to targeted genome modulation/manipulation is achieved through a variety of nucleotide sequence-specific binding proteins such as leucine zippers, zinc finger proteins, etc. These proteins are involved with gene regulation as transcription factors and/or can be used to induce DSB at a native genomic location. The DSB can be provided by several different classes of sequence-specific nucleases such as meganucleases, leucine zippers, zinc finger proteins, etc., and, more recently, the development of novel chimeric versions of these proteins. One of the best described nucleotide-specific binding proteins are the Zinc Finger Proteins (ZFP). The C2H2 zinc finger was discovered in the amphibian transcription factor TFIIIA, and has since been found to be the most common DNA recognition motif in all species of metazoa. The X-ray crystal structure of the C2H2 ZFP, Zif268, revealed a strikingly syllabic mode of protein-DNA recognition, with each zinc finger specifying a 3 or 4 bp subsite in the context of a tandem arrangement, and suggested the possibility of using this peptide motif as a scaffold for DNA binding domains with novel specificities. Since then, a large number of ZFPs engineered to bind novel sequences have been successfully used in many different laboratories in the context of artificial transcription factors and other functional chimeric proteins. The C2H2 zinc finger protein domain has been used as a scaffold for sequence-specific DNA binding (Pavelitch and Pabo (1991) Science 252:809-17) and ZFNs produced by fusing zinc finger protein domains with a sequence-independent nuclease domain derived from the Type IIS restriction endonuclease FokI (Kim et al. (1996) Proc. Natl. Acad. Sci. USA 93(3):1156-60). Engineered ZFNs have been used to drive high-efficiency targeting to an endogenous genomic locus in transformed (Moehle et al. (2007) Proc. Natl. Acad. Sci. USA 104(9):3055-60) and primary human cells (Lombardo et al. (2007) Nature Biotechnol. 25:1298-306).
Initial attempts at using ZFNs in plants have been promising (Lloyd et al. (2005) Proc. Natl. Acad. Sci. USA 102(6):2232-7; Wright et al. (2005) Plant J. 44(4):693-705; Maeder et al. (2008) Mol. Cell 31(2):294-301). A construct carrying a ZFN gene under the control of an inducible promoter along with its corresponding recognition sequence was stably integrated into Arabidopsis and shown to introduce targeted mutations resulting from non-homologous end joining at the recognition site at frequencies averaging 7.9% among induced progeny seedlings (Lloyd et al. (2005), supra). Similarly, among 66 tobacco plants regenerated from protoplasts transformed with a ZFN designed to cleave at the SuRA locus, three displayed single base pair deletions at the target site resulting from non-homologous end joining repair (Maeder et al. (2008), supra). Tobacco cells, containing a pre-integrated, non-functional reporter gene missing 600 bp directly flanking a zinc finger recognition sequence, when co-transformed with constructs containing a corresponding ZFN gene and donor DNA homologous to the pre-integrated sequence comprising the missing 600 bp, showed evidence of homology-directed repair of the reporter gene (Wright et al. (2005), supra). Most recently, a yeast-based assay was used to identify ZFNs capable of cleaving a plant endochitinase gene (Cai et al. (2009) Plant Mol. Biol. 69(6):699-709). Agrobacterium delivery of a Ti plasmid harboring both the ZFNs and a donor DNA construct comprising a pat herbicide resistance gene cassette flanked by short stretches of homology to the endochitinase locus yielded up to 10% targeted, homology-directed transgene integration precisely into the ZFN cleavage site. It is important to note that other zinc finger designs based on a C3H1 design have been demonstrated in plants (Shukla et al. (2009) Nature 459:437-41, Cai et al. (2009), supra).
The present invention relates to methods using nanoparticles to non-invasively deliver sequence-specific nucleases into plant cells having a cell wall.